Cell dispersal method for monkey kidney tissue

ABSTRACT

A method for increasing the cell yield from mammalian kidney tissue by perfusion of the decapsulated kidney in situ with a proteolytic enzyme under pressure and a means for dispersal of the cells by mechanical agitation.

United States Patent Daly et al.

[54] CELL DISPERSAL METHOD FOR MONKEY KIDNEY TISSUE [72] Inventors: William F. Daly, Hillsdaie; Ronald J. Vallancourt, Park Ridge, both of [73] Assignee: American Cyanamid Company,

Stamford, Conn.

[22] Filed: May 17,1971

[21] Appl.No.: 144,405

521 u.s.c1. ..-....12s/1n,195/1.8 511 1111.01. .A61b 19/00 [58] FieldofSearch ..12s/1 R;l95/1.7,1.8, 127

[56] References Cited I NITE STQTESPAEN' EW V.

2,958,517 11/1950 m ke:ez 1,..... .,.,.195/17 11s] 3fl2fi05 1 Nov. M, 1972 Primary Examiner-Dalton L. Truluck Attorney-Stephen Raines [57] ABSTRACT A method for increasing the cell yield from mammalian kidney tissue by perfusion of the decapsulated kidney in situ with a proteolytic enzyme under pressure and am'eans for dispersal of the cells by mechanical agitation.

CELL DISPERSAL METHOD FOR MONKEY KIDNEY TISSUE BACKGROUND OF THE INVENTION In the preparation of vaccines, cells from kidney tis- I sue are frequently used for culturing a virus, such as monkey kidney tissue for the poliomyelitis virus. Prior to this invention, cells of kidney tissue origin were obtained by removal of the kidney prior to treatment with a trypsin solution to digest unwanted intercellular material or by introducing a trypsin solution by way of the descending aorta or left ventricle into a monkey overdosed with an anesthetic such as phenobarbital, ether, etc. and allowing it to flush by gravity flow through the kidney in situ. The expense required in maintaining large animal colonies, carryingout surgical techniques on animals, and separating the kidney tissue cells is very considerable. In an effort to minimize this expense, the pharmaceutical industry has searched for methods to maximize the yield of kidney tissue cells obtained from each animal, thus enabling the maintenance of smaller animal colonies and to minimize the' surgical and separation procedures.

SUMMARY OF THE INVENTION The present invention provides a method for increasing the yield of mammalian kidney tissue cells, more specifically, monkey, rabbit, and dog kidney tissue cells, by perfusion of the kidneys, preferably in their decapsulated state, under pressure and controlled temperatures. The perfusion is carried out using a calculated volume of proteolytic enzyme, preferably trypsin, in a suitable solution for injection which may contain components such as buffers, preservatives, etc. Other useful proteolytic enzymes are pancreatin, collagenase,

etc. This solution is introduced under pressure directly into the vascular system of the mammal, preferably into the renal artery and is maintained at a constant temperature, at or above room temperature during the procedure. The renal veins and ureters are severed and renal capsule removed to permit rapid drainage of the spent enzyme solution. Following this the kidneys are removed and cell dispersal accomplished by mechanical stirring. No additional proteolytic enzyme is required following the in situ perfusion.

In accordance with this invention, a suitable mammal is operated upon utilizing the accepted techniques designed to clear the surgical area. The kidneys may be isolated by severing any matter connected to them except for the renal arteries and veins. This includes severing of the ureter to each kidney. One or more punctures are made in the abdominal aorta at or near the point of emergence of the renal arteries and cannulae inserted through the puncture or punctures into both renal arteries. A buffered solution of a proteolytic enzyme is introduced under a pressure of just above 0 to 15 lbs. per sq. in. The concentration of the proteolytic enzyme may vary slightly. The volume of solution can be varied from about 300 milliliters to about 1000 milliliters. When trypsin is employed, satisfactory results were obtained with 600 milliliters of a solution of about 0.25 percent of a 1:250 grade trypsin in buffered saline solution, although volumes of about 1000 ml. could also be employed. It may be noted that 1:250 grade trypsin gave somewhat better results than pure trypsin. The renal veins and ureters are severed to permit rapid drainage of the spent trypsin, and the renal capsules removed to allow for both drainage and expansion of the kidneys. After the perfusion is completed, the remaining vessels (renal arteries) are severed and the kidneys are removed, placed in a suitable container and the tissue from the kidneys is mechanically stirred in a growth medium, such as Mel- .nicks, Eagles BME, Eagle's MEM, Morgan's 199, or

Hanks Lactal, until cell dispersal is complete. Gross particles may be removed by filtration. While additional trypsin may be added to further effect the digestion of intercellular material, it is not considered necessary.

The cells upon completion of the dispersion are suitable for in vitro cultivation (Table V).

While the most significant improvement over earlier methods of cell dispersal for kidney tissue is the use of pressure, removal of .the renal capsule, injecting the enzyme solution directly into the renal arteries and cutting the renal veins, numerous other improvements have been incorporated in order to optimize yields. These additional improvements consist of the followmg:

a. Utilization of a temperature of about 36 C. to 38 b. The use of a technique resulting in the slow exsanguination of the animal.

c. Utilization of a technique which caused the pressure to be uniformly decreased, that is in contrast to a pulsating type pressure.

d. Cell dispersal accomplished through only mechanical agitation following the in situ perfusion. Trypsin not being necessary in this step.

While relying on a technique that employs introducing the solution under pressure into a kidney in which the renal capsule has been removed with less than all the modifications one obtains to 200 percent increases'in yields of viable cells (Table II) over. earlier techniques. However, the ideal conditions which incorporate all of the aforementioned modifications raise the yields even more significantly (Table Ill). Thus depending upon equipment available, objectives, etc.; one may incorporate some or all of the modifications described by Applicants in practicing Applicants invention.

Table IV summarizes cell yields wherein one kidney was perfused and the other not perfused.

Table V summarizes the results when four monkeys intended for polio virusproduction were studied. In each case, one kidney was perfused and one kidney was not perfused.

DETAILED DESCRIPTION OF THE PREFERRED ENIBODIMENT EXAMPLE I A series of 24 monkeys (rhesus and cercopithecus) were prepared for surgery. That is, the monkeys were anesthetized, hair was removed from all but the head, tail and lower legs, the animals were washed with surgical soap and the hairless areas were painted with an iodine solution. The above procedure renders the operating field free of extraneous material which could cause contamination.

Each animal was placed head down, with its body at a 45 angle. The exsanguination of the animal took place slowly, usually by cardiac puncture. The entire operative field was swabbed with an alcohol solution and covered with a self adhesive, sterile, antistatic plastic disposable drape such as steri-drape 1050 (Minnesota Mining and Mfg. Co.). Starting in close proximity to the clavicle, a skin incision was made caudally over the thoracic area along a line midway between the spinal column and the sternum continuing over the abdomen along a line midway between the spinal column and the linea alba and extending to the groin region. The incision crosses tranversely just anterior to the pelvis to the opposite side of the monkey where it is then directed cranially following a similar course. The skin was now removed and reflected cranially. The abdominal cavity was opened at a point above the urinary bladder. The abdominal musculature was cut, following the same course as the skin incision. The diaphragm was punctured and the ribs and intercostal muscles were cut. The diaphragm and mediastinal attachments to the sternum were severed allowing all structures mentioned to be reflected cranially or removed completely thus exposing the heart and lungs. The descending colon was looped outside the abdominal cavity. The attachment of the mesentery to the colon (descending and transverse) was severed at a point in close proximity to the bowel wall. The gastrorenal ligament was severed thus freeing the right kidney from its attachment to the stomach. The attachments of the ascending colon and caecum to the dorsal body wall were severed and the bowel was reflected cranially. The remnant of the right diaphragm was cut along the muscular attachment to the ribs. The posterior vena cava and the abdominal aorta were exposed and clamped off just anterior to their bifurcation into the common iliac arteries and veins. The posterior vena cava was bisected anterior to the hemostat and was freed from its fibrous attachments up to the point of entry of the renal veins. A hemostat was applied to the bisected end of the vein. The phrenicolienel ligament was severed and the spleen and pancreas reflected cranially. The remnant of the left diaphragm was cut along the muscular attachment to the ribs. The thoracic aorta was clamped at a point midway between the heart and the vestigial aortic hiatus.

The adrenal glands were dissected from the cephalic pole of the respective kidneys. The fibrous band of tissue connecting the kidneys to the dorsal body wall was severed. The ureters were exposed and bisected. Each kidney was now held to the body wall by the respective renal arteries and veins only. The abdominal aorta and the renal arteries were exposed. A puncture was made in the aorta at the point of emergence of the renal arteries. Cannulae of suitable bore size (20-22 gauge) and length (one-fourth inch to 1 inch) were inserted through the punctured aorta and into the left and right renal arteries. To insure that the cannulae are not forced out of the arteries when pressure is applied during the procedure, a ligature was placed around each artery at the point of emergence from the aorta. Plastic tubing was connected to each cannula leading to a plastic bag such as is used for the collection and/or storage of blood or blood products containing 600 ml. of 0.25 percent trypsin solution (1:250 grade trypsin in phosphate buffered saline, Dulbecco) at 21 43 C. It proved advantageous'to maintain the temperature at 37.8C. One procedure used was to immerse the plastic bags in a water bath. An initial pressure of 0-6 p.s.i. was applied to each plastic bag. A conventional hand press was one method used. A second method was to place metal weights on the plastic bags. The renal veins were immediately severed to permit drainage of the spent trypsin and the renal capsule was stripped from each kidney to allow for expansion. The procedure was containued for an average of 30 35 minutes until the 300 ml. to 1000 ml. of trypsin solution had perfused each kidney. Each pair of kidneys were removed by standard technique and placed in a flask containing 300 ml. of a buffered growth medium with added lactal albumin hydrolysate and calf serum, such as Melnicks Medium A." Continuous mechanical stirring was performed until cell dispersal was complete (approximately 15 minutes). The cell suspension was transferred to a cell collecting flask through fine stainless steel mesh to remove gross particles. A sample was removed for the viable cell count. Viable cell counts were made using the method of dye exclusion (Trypan blue) as described by Pappenheimer, Jr. Experimental Medicine 25, 633 (1917), and McLemans, Jr. Bact. 74, 768 (1957). The results appear in Tables 1- V.

% lncrease== 14.52

TABLE II Experimental Conditions (Room Temperature [21-32] Pulsating pressure, cut veins and cut ureters) Type of Millions of Percent Increase Monkey Viable cells/Monkey Over Control Rhesus 636 98.8

537 67.8 Cercopithecus 684 1 13.8

501 56.6 Rhesus 531 65.9

492 53.8 Cercopithecus 525 64. l

6 513 60.3 630 96.9 537 67.8 705 120.3 660 106.2 Rhesus 711 122.2 675 110.9 906 183.1 459 43.4 714 123.1 Rhesus 468 46.2 750 134.4 1014 220.1 5 825 157.8 900 181.3 765 139.1 816 155.0 Av. 657 Av. 105 1 3% 122 Control 320 0 72o b 10 630 96.9 810 153. l 750 134.4 The control for this experiment consisted of 100 monkeys where kid- 936 192.5 neys were processed by the method of Youngner, J.S., Proceedings of 870 171.9 the Society for Experimental Biology and Medicine 85, 202-205 (1954), 954 198.1 and Bodian, D., Virology, 2, 575 (1956), and for which the average 627 95.9 yield of viable cells per monkey was 320 million. 1 5 948 196.3 750 134.4 747 133.4 TABLE 111 v 648 102.5 651 103.4 11 .465 45 3 603 88.4 510 59.4 795 148.4 Expenmental Conditions 11 915 185,9 909 184.1 Temp. 36-3 8 C.- Uniform Decreasing Pressure (3.5 I: 32g psi to 0 psi) Slow exsanguination and rapid transfer to a 161 5 owth media. 660 106-3 gr Y 723 T of Millions of Percent Increase I. 11014 M rfkey Viable cells/Monkey Over 0611661 u 28g 843 163 4 936 192.5 11 76 139. Rhesus 720 125.0 30 4 69(5) 1 879 174-7 11 1,473 360.3 666 103-1 11 1,068 233.8 633 11 1,095 242.2 11 855 167.2 834 1606 11 720 125.0 528 65.0 50 94 699 109.1 u 59 1175 480 50.0 11 33 973 723 125.9 11 v 744 1315 534 11 666 108.1 690 115-6 11 795 148.4 645 101.6 780 143.8 4 6313 Cercopithecus 621 94.1 930 190.6 855 167.2 879 174.7 11 714 123.1 750 134.4 837 161.6 924 188.8 960 200.0 924 188.8 999 212.2 798 149.4 900 181.3 11 684 1 1 Ce'reopithecus 807 152.2 522 63.1 759 7 137.2 837 1 1 810 153.1 504 57.5 681 112.8 11 726 126.9 11 1,002 213.1 11 648 102.5 11 765 139.1 11 858 168.1 11 729 127.8 798 149.4 11 843 163.4 798 149-4 687 1 14.7 978 205.6 786 145.6 11 1 216.9 588 83.8 768 140.0 1,143 257.2 702 1 19.4 663 107.2 612 91.3 816 155.0 11 846 164.4 11 1,260 293.8 11 1128 252.5 11 492 53.8 11 594 85.6 11 684 1 13.8 459 43.4 558 74.4 639 99.7 915 185.9 924 188.8 774 141.9 573 79.1 738 130.6 795 148.4 813 154.1 921 187.8 1,035 223.4 636 98.8 966 201.9 834 160.6 633 97.8 11 768 140.4 11 804 151.3 834 160.6 735 129.7 11 885 176.6 11 810 153.1 11 997 21 1.6 11 591 84.7 37 804 151.3 11 609 90.3 11 441 37.8 11 729 127.8 693 1 16.6 630 96 9 Av. 770 Av. 141

TABLE IV Million Viable Cell per Kidney Percent lncrease Type of Monkey A B by Perfusion Cercopithecus 265 l 60 66 Cercopithecus 152 128 19 Cercopithecus 149 82 82 Rhesus 296 27 l 9 Rhesus 181 79 129 Rhesus 205 118 74 Rhesus 238 93 156 Average 2 1 2 l 33 76 A Perfused B Non-Perfused TABLE V Million Process Viable Cell Method Cells/ Cultures Control Used Kidney Prepared Growth (24 days) Non- Perfused 296 19 Good Fair Non-Perfused 296 19 Good Fair Perfused 305 21 Good Good Non-Perfused 316 21 Good Good Perfused 287 19 Good Good Non-Pcrfused 300 21 Good Good Perfused 404 28 Good Good Non-Perfused 137 Good Good Perfused 165 1 3 Good Fair We claim:

1. A method for increasing the yield of mammalian kidney tissue obtained from a surgically prepared mammal having each kidney held to the body wall by the respective renal arteries and veins, (in situ) which comprises: inserting and securing cannulae into said renal arteries of said mammal; ligating the renal arteries; perfusing said kidneys with a solution introduced into said cannulae containing a proteolytic enzyme under pressure; at a temperature of 21 to 43 C. and removing a capsule from said kidneys permitting expansion caused by said solution containing a proteolytic enzyme.

2. A method as recited in claim 1 in which said ureter connected to said kidney is severed to permit rapid drainage of the spent proteolytic enzyme solution.

3. A method as recited in claim 2 in which said renal veins connected to said kidney is severed to permit rapid drainage of the spent proteolytic enzyme solu- 2. A method as recited in claim 3 wherein said mammal is a species of monkey.

5. A method as recited in claim 4 wherein said solution containing a proteolytic enzyme is a trypsin 1:250 solution. I

6. A method as recited in claim 5 wherein said pressure is above 0 1b./sq. in. to about 15 lbs/sq. in. and is uniformly diminished during perfusion.

7. A method as recited in claim 6 wherein said temperature is 363 8 C.

8. A method as recited in claim 6 wherein said solution containing said trypsin also contains components selected from the group consisting essentially of preservatives, bufiers, and inorganic salts.

9. A method as recited in claim 8 wherein about 600 milliliters of said solution having about 0.25% w/v concentration of trypsin (1:250 grade trypsin in buffered saline) is used during the perfusion.

10. A method as recited in claim 9 wherein said kidney is removed after perfusion and by continuous mechanical stirring in a growth medium a uniform dispersion of kidney cells is obtained which is suitable for classic tissue culture used.

11. A method as recited in claim 2 wherein said kidney is removed after perfusion and only by continuous mechanical stirring is transformed into a cell dispersion. 

2. A method as recited in claim 1 in which said ureter connected to said kidney is severed to permit rapid drainage of the spent proteolytic enzyme solution.
 3. A method as recited in claim 2 in which said renal veins connected to said kidney is severed to permit rapid drainage of the spent proteolytic enzyme solution.
 4. A method as recited in claim 3 wherein said mammal is a species of monkey.
 5. A method as recited in claim 4 wherein said solution containing a proteolytic enzyme is a trypsin 1:250 solution.
 6. A method as recited in claim 5 wherein said pressure is above 0 lb./sq. in. to about 15 lbs./sq. in. and is uniformly diminished during perfusion.
 7. A method as recited in claim 6 wherein said temperature is 36*- 38* C.
 8. A method as recited in claim 6 wherein said solution containing said trypsin also contains components selected from the group consisting essentially of preservatives, buffers, and inorganic salts.
 9. A method as recited in claim 8 wherein about 600 milliliters of said solution having about 0.25% w/v concentration of trypsin (1:250 grade trypsin in buffered saline) is used during the perfusion.
 10. A method as recited in claim 9 wherein said kidney is removed after perfusion and by continuous mechanical stirring in a growth medium a uniform dispersion of kidney cells is obtained which is suitable for classic tissue culture used.
 11. A method as recited in claim 2 wherein said kidney is removed after perfusion and only by continuous mechanical stirring is transformed into a cell dispersion. 